Method for Hematology Analysis

ABSTRACT

A method whereby one or more fluorescent dyes are used to bind and stain nucleic acids in certain blood cells, such as, for example, white blood cells, nucleated red blood cells, and reticulocytes, and to induce fluorescent emissions upon excitation of photons from a given source of light, such as, for example, a laser, at an appropriate wavelength. More particularly, this invention provides a method whereby a fluorescent trigger is used in a data collection step for collecting events that emit strong fluorescence, in order to separate white blood cells and nucleated red blood cells from red blood cells and platelets without the need for using a lysing agent.

RELATED APPLICATIONS

The present application claims priority from of co-pending U.S.Provisional Patent Application No. 61/331,516, filed May 5, 2010 andco-pending U.S. Provisional Patent Application No. 61/331,867, filed May6, 2010.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for hematology analysis, whereinfluorescent dyes are used to distinguish various components of a sampleof blood.

2. Discussion of the Art

The CELL-DYN® Sapphire™ automated hematology analyzer, as well as theCELL-DYN® 4000 automated hematology analyzer, both of which arecommercially available from Abbott Laboratories, Santa Clara, Calif.,are equipped with an optical bench that can measure multi-angle lightscatter and fluorescence, as described in U.S. Pat. Nos. 5,631,165 and5,939,326, both of which are incorporated herein by reference.Furthermore, U.S. Pat. Nos. 5,516,695 and 5,648,225, both of which areincorporated herein by reference, describe a reagent suitable for lysingred blood cells and staining nuclear DNA of membrane lysed erythroblaststo discriminate white blood cells from erythroblasts. Membrane lysederythroblasts are erythroblasts wherein the membrane thereof hasundergone lysis. U.S. Pat. No. 5,559,037, incorporated herein byreference, describes the simultaneous detection of erythroblasts andwhite blood cell differential by means of a triple triggering circuitry(AND/OR), which is used to eliminate noise signals from cell debris,such as, for example, membranes of lysed red blood cells, which arelocated below the lymphocyte cluster along the Axial Light Loss (ALL)axis of a cytogram. However, the use of lysing agents to lyse red bloodcells brings about certain difficulties and complications in thedetection of red blood cells and white blood cells. The lysing agent maybe insufficiently strong, thereby resulting in red blood cells beingcounted as white blood cells. Alternatively, the lysing agent may beexcessively strong, thereby resulting in artificially low counts ofwhite blood cells. Different samples require lysing agents of differentstrengths in order to obtain accurate counts of white blood cells;accordingly, all hematology analyzers currently in use sometimes yieldincorrect counts of white blood cells.

In hematological assays aimed at determining parameters from human wholeblood, there are two physiological factors that present obstacles tosimple, rapid, and accurate determination of cell counts. One factor isthat, in typical fresh peripheral human whole blood, there are about1,000 red blood cells and about 50 platelets for each white blood cell.The other factor is that, while platelets are typically sufficientlysmaller than any other cell type to allow discrimination based on size,and most white blood cells are sufficiently larger than either red bloodcells or platelets to again allow discrimination based on size, two cellspecies in particular—red blood cells and lymphocytes, a subtype ofwhite blood cells—typically overlap in size distribution (as well as intheir scattering signatures) to a sufficient degree to makediscrimination based on size prone to gross error. Therefore, whendetermining red blood cells mainly by size discrimination, the asymmetryin concentration works in one's favor, since the occasional white bloodcell misclassified as a red blood cell will not, generally, affect theoverall accuracy of the measured concentration of red blood cells to anyappreciable degree; however, the converse is not true, and anyunaccounted for interference from red blood cells in determining theconcentration of lymphocytes (and, by extension, the overallconcentration of white blood cells) would yield very inaccurate results.

Consequently, methods have been developed in the prior art to handlethis large asymmetry and size overlap and still provide useful resultsin an acceptable time frame. One standard method employed in the priorart has been to separate the blood sample to be analyzed into at leasttwo aliquots, one destined for red blood cell and platelet analysis, andone for white blood cell analysis. The aliquot destined for white bloodcell analysis is mixed with a reagent solution containing a lysingreagent that preferentially attacks the membranes of the red bloodcells. Partially on account of their loss of hemoglobin through thecompromised membrane, and partially on account of their attendantreduction in size, the resulting lysed red blood cells becomedistinguishable from lymphocytes based on their respective scatteringsignatures. Another method employed in the prior art involves usingnucleic acid dyes to provide a fluorescent distinction between the redblood cells and the white blood cells. White blood cells contain anucleus containing DNA. When these white blood cells are labeled via afluorescent label, they can be distinguished from mature red bloodcells, whose nuclei have been expelled in the maturation process.

Both of these methods have drawbacks. First of all, the lysing reagentused to dissolve the red blood cells can attack the white blood cells aswell, reducing their integrity and eventually dissolving them. Thisdrawback is magnified with fragile white blood cells, which are abnormalon account of some type of pathological condition (such, as, forexample, chronic lymphocytic leukemia). Another drawback is attributableto certain types of red blood cells, such as, for example, those foundin neonates, and in patients with thalassemia, sickle-cell anemia, andliver disease, which red blood cells are naturally resistant to lysis,and which red blood cells therefore tend to persist as interferents inwhite blood cell assays involving lysis. In order to reduce thelikelihood of either degradation of white blood cells or interferencefrom unlysed red blood cells (either of which would jeopardize theaccuracy of the overall white blood cell concentration measurement), acarefully selected combination of a lysing agent, concentration of thelysing agent, control of temperature, and time of incubation must beused. In some cases, the user is offered several test options withdifferent lysing conditions, thereby allowing the user to tailor theassay to the subject patient sample. This tailoring, however, is acomplex solution, which additionally either requires prior knowledge ofthe state of the patient, or must be used as a reflex test following astandard complete blood count (CBC).

Turning to the previously mentioned fluorescence-based approach fordiscriminating red blood cells from lymphocytes, a major obstacle is themeasurement rate. When white blood cells are measured at the same timeas red blood cells and platelets, the presence of red blood cells setsan upper limit to the concentration that can be sent through theanalyzer without incurring in coincidences at an unacceptably high rate;the dilution ratio used to achieve such concentration, in turn, limitsthe rate at which white blood cell events are being counted; and inorder to obtain the counting precision expected of the analyzer, thisrelatively low rate of white blood cell event acquisition, in turn,results in long acquisition times. For example, the concept of measuringall of the components of blood from a single sample in one pass wasdisclosed in U.S. Pat. No. 6,524,858. As noted in that disclosure, themethod would be capable of a cycle time of 88 seconds, or about 41CBC/hr. This throughput is far lower than that achievable by mostautomated hematology analyzers commercially available today, severelylimiting the commercial usefulness of the one pass method.

The CELL-DYN® Sapphire™ hematology analyzer, as another example,presently offers a test selection (requiring yet another aliquot ofsample in addition to those used in the red blood cell/platelet assayand in the white blood cell assay) employing a nucleic-acid dye capableof differentiating between red blood cells and lymphocytes. This testselection uses the dye primarily to differentiate between mature redblood cells and reticulocytes, a subset of immature red blood cells thatretain dye-absorbing RNA in the cytoplasm. While it would technically bepossible to count the white blood cells using this same assay, becausethey are sufficiently differentiated by fluorescence from either redblood cells or reticulocytes to obtain the desired accuracy, therelatively low concentration of white blood cells in the dilution usedmakes it an impractical option to achieve the required statisticalprecision. Such a scheme would require an acquisition time ofapproximately 75 seconds, limiting throughput to only 48 CBC/hr.Accordingly, although this approach is theoretically feasible, a muchhigher throughput would be required in order for this approach to becomepractical commercially.

Although modern five-part differential hematology analyzers are capableof reporting more hematology parameters, and consequently, providingmore useful diagnostics information, almost all of them containsophisticated fluidic systems, reagent systems, and hardware systems inorder to facilitate a number of different assays on blood samples ofpatients. The complex design often results in higher overall costs ofhematology analyzers, as well as greater possibility of poor reliabilityof the hematology analyzers.

Red blood cells and platelets, as well as their associated parameters,are measured by means of impedance or optical methods, following adilution of the blood sample with diluent. Quantification of hemoglobinrequires lysis of red blood cells by means of a mixture of hemoglobinlysing reagent and, in most cases, a diluent. White blood cell count andwhite blood cell differential analysis rely on a separate reagent orreagents for lysing red blood cells, minimizing red blood cellfragments, and stabilizing white blood cells for differentialmeasurement. Additional reagents, which may contain one or morefluorescent dyes, are needed to allow hematology analyzers to conductmore complete analysis of blood cells, including reticulocytes andnucleated red blood cells.

Therefore, it would be desirable to develop a method for identifying,analyzing, and quantifying the cellular components of a sample of wholeblood by means of multi-angle light scatter without the need for lysingred blood cells, complex fluidic systems, complex reagent systems, orcomplex hardware systems.

SUMMARY OF THE INVENTION

This invention provides a method whereby one or more fluorescent dyesare used to bind and stain nucleic acids in certain blood cells, suchas, for example, white blood cells, nucleated red blood cells, andreticulocytes, and to induce fluorescent emissions upon excitation ofphotons from a given source of light, such as, for example, a laser, atan appropriate wavelength. More particularly, this invention provides amethod whereby a fluorescent trigger is used in a data collection stepfor collecting events that emit strong fluorescence, in order toseparate white blood cells and nucleated red blood cells from red bloodcells and platelets without the need for using a lysing agent.

In one embodiment, the method employs a plurality of optical channelsand at least one fluorescent channel for collecting data and analyzingthe data in order to identify each cell population and reveal additionalinformation relating to a sample of blood.

The method described herein can reduce the complexity of hematologyanalyzers, which analyzers would require no more than two reagents forcarrying out all hematology assays, including assays for determiningreticulocytes and nucleated red blood cells. In addition, the fluidicscomponents and hardware components, and consequently, overall cost, canbe greatly reduced. More importantly, the error frequency of thehematology analyzer can also be greatly reduced. The lysis-free approachdescribed herein eliminates the adverse effects of a lysing agent forred blood cells on samples of blood, including samples of bloodcontaining lyse-resistant red blood cells and fragile white blood cells.It should be noted, however, that the method described herein can alsobe used when red blood cells undergo complete lysis or partial lysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating the illumination anddetection optics of an apparatus suitable for generatingthree-dimensional signals from cells for differential analysis.

FIG. 2 illustrates a scheme for aiding in setting a fluorescent trigger.The horizontal scale represents a logarithmic scale ranging from a valueof 1 to a value of 1000 for values of fluorescence. The values arearbitrary units.

FIG. 3 is a cytogram illustrating the separation of major cellpopulations of a sample of whole blood, i.e., red blood cells (RBC),platelets (PLT), and white blood cells (WBC) by means of axial lightloss (ALL) and intermediate angle scatter (IAS) of light. Noise is alsodepicted.

FIG. 4 is a cytogram illustrating the further separation ofreticulocytes (Retics) from mature red blood cells (RBC) by means offluorescence, following interaction of a fluorescent dye and RNA.

FIG. 5 is a plot illustrating the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for red blood cells (RBC). The symbol M in theconcentration scale represents 1×10⁶.

FIG. 6 is a plot illustrating the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for platelets (PLT). The symbol K in the concentrationscale represents 1×10³.

FIG. 7 is a cytogram illustrating separation of fluorescent (bright)platelets (PLT) from white blood cells (WBC) of a sample of whole bloodby means of axial light loss (ALL) and intermediate angle scatter (IAS)of light.

FIG. 8 is a cytogram illustrating the three major subpopulations ofwhite blood cells, namely, neutrophils (NE), lymphocytes (LY), andmonocytes (MO) by means of polarized side scatter (PSS) and axial lightloss (ALL). FIG. 8 is in a dot plot format.

FIG. 9 is a cytogram illustrating the three major subpopulations ofwhite blood cells, namely, neutrophils (NE), lymphocytes (LY), andmonocytes (MO) by means of polarized side scatter (PSS) and axial lightloss (ALL). FIG. 9 is in a contour plot format. FIG. 9 is based on thesame white blood cell data as was used in FIG. 8.

FIG. 10 is a cytogram illustrating the separation of eosinophils (EO)from the remaining white blood cells by means of depolarized (DSS) andpolarized side scatter (PSS).

FIG. 11 is a plot illustrating the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for white blood cells (WBC). The symbol K in theconcentration scale represents 1×10³.

FIG. 12 is a plot illustrating the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for neutrophils.

FIG. 13 is a plot illustrating the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for lymphocytes.

FIG. 14 is a plot illustrating the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for monocytes.

FIG. 15 is a plot illustrating the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for eosinophils.

FIG. 16 is a cytogram illustrating a method of distinguishing nucleatedred blood cells (NRBC) from white blood cells.

FIG. 17 is a cytogram illustrating a white blood cell differentialanalysis wherein neutrophils (NE), monocytes (MO), and lymphocytes (LY)are separated from the other white blood cell components.

DETAILED DESCRIPTION

As used herein the expression “axial light loss” and “ALL” refer to themeasurement of the total light lost from the laser beam at from about 0°to about 1° when a particle passes through the beam. This parameterrelates to measurement of light extinction, i.e., light lost throughscattering as well as through absorption; in the absence of absorption,“axial light loss” is a measurement that correlates broadly with thesizes of cells or particles passing through an optical detection system.As used herein, the expressions “intermediate angle scatter” and “IAS”refer to the measurement of forward light scatter at intermediate anglefrom 3° to 10°. This parameter relates to measurement of complexity of acell. As used herein, the term “complexity” refers to the composition ofa cell. Some cells have mitochondria, ribosomes, nucleus, while othercells lack one or more of the foregoing components. The measuredintensity of “IAS” depends to some degree on the heterogeneity of thecontents of a cell (or particle) passing through the illumination beamof a cytometer. The density of “IAS” signals can be thought of as ameasure of the complexity of the contents of the cell, i.e., thepresence of organelles, such as, for example, nuclei, vacuoles,endoplasmic reticula, mitochondria, etc. As used herein, the expressions“polarized side scatter” and “PSS” refer to polarized light scatter atthe angle of 90°. This parameter relates to measurement of lobularity.The nuclei of cells have various shapes that may result in one to fivelobules, inclusive. A representative example of a cell with multi-lobednucleus is a segmented neutrophil. As used herein, the expressions“depolarized side scatter” and “DSS” refer to depolarized light scatterat the angle of 90°. This parameter relates to measurement ofsubpopulations of blood cells. Blood cells have various numbers ofsubpopulations within the membranes of the cell. Examples of thesesubpopulations, for white blood cells, are eosinophils, neutrophils,basophils, monocytes and lymphocytes

As used herein, the term “trigger” means the minimum value of a signalthat a measurement of the signal must exceed to be considered valid.Events providing a signal value above the trigger value are collectedand recorded. Events providing a signal value below the trigger valueare ignored. With respect to the trigger values used herein, a scale of0 to 5000 or a scale of 0 to 6000 is used for each channel of detection.Each value on the scale represents a gradation of the strength of asignal, with the value of 0 being the lowest strength and the value of5000 (or 6000) being the highest strength. In FIG. 2, the scale istruncated at the value of 1000.

As used herein, the term “erythroblast” means any of the nucleated cellsin bone marrow that develop into erythrocytes. As used herein, the term“erythrocyte” means the yellowish, non-nucleated, disk-shaped blood cellthat contains hemoglobin and is responsible for the color of blood. Asused herein, the expression “bright platelet” means a platelet thatemits a relatively strong fluorescent signal. Bright platelets arecollected in the white blood cell data block.

One or more detectors are preferably placed in the light path formeasuring forward intermediate angle scattering (IAS) and either smallangle forward scattering (SAS) or axial light loss (ALL). The light lossis generally due to scattering and defined as the decrease in lightenergy reaching a detector in the path of a laser beam due to thepassage of a cell through that beam. Generally ALL is detected at anangle of from about 0° to about 1°. SAS is light energy that reaches adetector outside, but within a narrow angle of about 1° to about 3°, theincident laser beam due to scattering from a cell passing through thebeam. A beam stop is generally provided to keep the laser beam fromgetting into the detector. ALL measuring systems collect light withinthe incident cone of laser illumination, while small angle scattersystems collect light outside this cone. In ALL measuring systems, thesignal of interest is a negative signal subtracted from the steady statelaser signal, whereas in the small angle forward scatter measurement,the signal is a small positive signal imposed on a very low backgroundlight level. Intermediate angle forward scattering (IAS) is similar tosmall angle forward scattering, except the light is scattered at alarger angle from the incident laser beam. More specifically, IASrelates to light scattered in a ring between 3° and 10° away from theincident or centerline of a laser beam. In a preferred embodiment, ALLis collected in the angles less than 0.3° horizontally and less than1.2° vertically from the laser axis, and IAS is collected at anglesbetween 3° and 10° from the laser axis.

As used herein, the expression “Open Mode” means that the sample ispresented directly to the automated instrument by a human operator. Asused herein, the expression “Closed Mode” means that the sample ispresented directly to the automated instrument by a robotic mechanism.

As used herein, the expression “measuring cells” refers to enumeratingcells by means of light scattering techniques to determine, for example,size, granularity, lobularity, and fluorescence when the cells arestained with a particular dye or fluorochrome.

The symbol “(s)” following the name of an object indicates that eitherthe object alone or a plurality of the objects is being referred to,depending upon the context of the statement surrounding the mention ofthe object or objects.

As used herein, the expression “morphological assessment” meansassessment of the shape of a cell. The term “leukocyte” means whiteblood cell. Unlike red blood cells, white blood cells occur in manydifferent types. Examples of leukocytes include granulocytes,neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Theexpression “reference method” means a method of the prior art againstwhich a test method is compared. The term “sickle cell” means a redblood cell shaped like a sickle. A sickle cell is typically resistant toa lyse reagent. The term “thalassemic” relates to a genetic blooddisorder in which the bone marrow cannot form sufficient red cells andred cell survival is also reduced. The term “lymphocyte” means a whiteblood cell that matures in lymph nodes, the spleen, and other lymphoidtissues, enters the blood, and circulates throughout the body. Theexpression “nucleated red blood cell” means an immature red blood cellthat still contains a nucleus. As used herein, the term “noise”includes, but is not limited to, such substances as lysed red bloodcells in particulate form, cell debris, and platelet clumps.

As used herein, the term “event” means a particle generating a signalthat is sufficient to trigger at least one detector, such as, forexample, the IAS detector, whereby that at least one detector signalsthe analyzer to collect measurements of that particle on all of thedetectors enabled on the analyzer, e.g., ALL, IAS, PSS, and DSS.Particles include, but are not limited to, are white blood cells (WBC),red blood cells (RBC), red blood cell fragments, platelets (PLT),lipids, platelet (PLT) clumps.

As used herein, the terms and phrases “diluent”, “sheath”, and“diluent/sheath”, and the like, mean a sheath diluent of the typesuitable for use with CELL-DYN® Sapphire™, CELL-DYN® Ruby™, CELL-DYN®3000 series, and CELL-DYN®4000 series hematology analyzers, which sheathdiluents are commercially available from Abbott Laboratories, SantaClara, Calif., and incorporated herein by reference.

As used herein, the term “data block” means data collected for a set ofcells having similar populations, with respect to the number of cellsper unit volume. For example, a given data block for a sample of bloodcan contain red blood cells (about 1×10⁶ red blood cells/μL),reticulocytes (about 1×10⁴ reticulocytes/μL), and platelets (about 1×10⁵platelets/μL). Another data block for a sample of blood can containwhite blood cells (about 1×10³ white blood cells/μL) and nucleated redblood cells (from about 10 to about 100 nucleated red blood cells/μL).

As used herein, the term “DNA” means deoxyribonucleic acid, which is apolymeric chromosomal constituent of living cell nuclei. As used herein,the term “RNA” means ribonucleic acid.

Automated hematology analyzers are discussed in WHITNEY WILLIAMS. Hem IAutomated Cell Counting and Evaluation. Educational publication[online], [retrieved on 2008 Jul. 15]. Retrieved from the Internet:<URL: http://www.clt.astate.edu/wwilliams/new_page_(—)4.html>,incorporated herein by reference. Representative examples of automatedhematology analyzers suitable for use herein include, but are notlimited to, CELL-DYN® Sapphire™ and CELL-DYN® Ruby™, modified byreplacing the laser thereof with a laser having a wavelength rangingfrom about 350 nm to about 700 nm. CELL-DYN® hematology analyzers arecommercially available form Abbott Laboratories, Santa Clara, Calif. Itshould be noted that any automated hematology analyzer based on theprinciple of flow cytometry can be modified to use a laser having awavelength ranging from about 350 nm to about 700 nm.

The essential components of systems of the prior art include a source oflight, a lens or system of lenses, a flow cell, and appropriatedetectors. In both the prior art and in the method described herein, thesources of light, the lens and the systems of lenses, the flow cells,and the detectors, and the functions thereof in a flow cytometry system,are well-known to those of ordinary skill in the art. See, for example,U.S. Pat. Nos. 5,017,497; 5,138,181; 5,350,695; 5,812,419; 5,939,326;6,579,685; 6,618,143; United States Patent Publication No. 2003/0143117A1, and U.S. patent application Ser. No. 12/767,611, filed Apr. 26,2010, and entitled METHOD FOR DISCRIMINATING RED BLOOD CELLS FROM WHITEBLOOD CELLS BY USING FORWARD SCATTERING FROM A LASER IN AN AUTOMATEDHEMATOLOGY ANALYZER, where sources of light, lenses, flow cells, anddetectors are described in greater detail. All of these references areincorporated herein by reference. See alsohttp://biology.berkeley.edu/crl/flow_cytometry_basic.html, Mar. 30,2006, pages 1-7, incorporated herein by reference. Lasers, lenses, flowcells, and detectors suitable for use in this invention are used incommercially available instruments from Abbott Laboratories, SantaClara, Calif., under the trademark CELL-DYN®.

The method described herein involves an automated method forsimultaneous analysis of white blood cell differential, erythroblasts,red blood cells, and platelets in liquid, such as, for example, blood.Other biological fluids, such as, for example, cerebrospinal fluid,ascites fluid, pleural fluid, peritoneal fluid, pericardial fluid,synovial fluid, dialysate fluid, and drainage fluid, can be used todetermine various parameters of these fluids.

Referring now to FIG. 1, an apparatus 10 comprises a source of light 12,a front mirror 14 and a rear mirror 16 for beam bending, a beam expandermodule 18 containing a first cylindrical lens 20 and a secondcylindrical lens 22, a focusing lens 24, a fine beam adjuster 26, a flowcell 28, a forward scatter lens 30, a bulls-eye detector 32, a firstphotomultiplier tube 34, a second photomultiplier tube 36, and a thirdphotomultiplier tube 38. The bulls-eye detector 32 has an inner detector32 a for 0° light scatter and an outer detector 32 b for 7° lightscatter.

In the discussion that follows, the source of light is preferably alaser. However, other sources of light can be used, such as, forexample, lamps (e.g., mercury, xenon). The source of light 12 can be avertically polarized air-cooled Coherent Cube laser, commerciallyavailable from Coherent, Inc., Santa Clara, Calif. Lasers havingwavelengths ranging from 350 nm to 700 nm can be used. A laser actuallyconstructed was a 407 nm laser purchased from Coherent. It should benoted that the laser was nominally designated to be a 405 nm laser, butthe actual wavelength of the laser was 407 nm. A custom made mountingplate was designed for this laser in order to be compatible with thecurrent optical bench of CELL-DYN® automated hematology analyzers.However, that particular mounting plate is not critical and othertechniques for mounting can be used. Operating conditions for the 407 nmlaser are substantially similar to those of lasers currently used withCELL-DYN® automated hematology analyzers.

Additional details relating to the flow cell, the lenses, the focusinglens, the fine-beam adjust mechanism and the laser focusing lens can befound in U.S. Pat. No. 5,631,165, incorporated herein by reference,particularly at column 41, line 32 through column 43, line 11. Thepreferred forward optical path system shown in FIG. 1 includes aspherical plano-convex lens 30 and a two-element photo-diode detector 32located in the back focal plane of the lens. In this configuration, eachpoint within the two-element photodiode detector 32 maps to a specificcollection angle of light from cells moving through the flow cell 28.The detector 32 can be a bulls-eye detector capable of detecting axiallight loss (ALL) and intermediate angle forward scatter (IAS). U.S. Pat.No. 5,631,165 describes various alternatives to this detector at column43, lines 12-52.

The first photomultiplier tube 34 (PMT1) measures depolarized sidescatter (DSS). The second photomultiplier tube 36 (PMT2) measurespolarized side scatter (PSS), and the third photomultiplier tube 38(PMT3) measures fluorescence emission from 440 nm to 680 nm, dependingupon the fluorescent dye selected and the source of light employed. Thephotomultiplier tube collects fluorescent signals in a broad range ofwavelengths in order to increase the strength of the signal.Side-scatter and fluorescent emissions are directed to thesephotomultiplier tubes by dichroic beam splitters 40 and 42, whichtransmit and reflect efficiently at the required wavelengths to enableefficient detection. U.S. Pat. No. 5,631,165 describes variousadditional details relating to the photomultiplier tubes at column 43,line 53 though column 44, line 4.

Sensitivity is enhanced at photomultiplier tubes 34, 36, and 38, whenmeasuring fluorescence, by using an immersion collection system. Theimmersion collection system is one that optically couples the first lens30 to the flow cell 28 by means of a refractive index matching layer,enabling collection of light over a wide angle. U.S. Pat. No. 5,631,165describes various additional details of this optical system at column44, lines 5-31.

The condenser 44 is an optical lens system with aberration correctionsufficient for diffraction limited imaging used in high resolutionmicroscopy. U.S. Pat. No. 5,631,165 describes various additional detailsof this optical system at column 44, lines 32-60.

The functions of other components shown in FIG. 1, i.e., a slit 46, afield lens 48, and a second slit 50, are described in U.S. Pat. No.5,631,165, at column 44, line 63 through column 45, line 26. Opticalfilters 52 or 56 and a polarizer 52 or 56, which are inserted into thelight paths of the photomultiplier tubes to change the wavelength or thepolarization or both the wavelength and the polarization of the detectedlight, are also described in U.S. Pat. No. 5,631,165, at column 44, line63 through column 45, line 26.

The photomultiplier tubes 34, 36, and 38 detect either side-scatter(light scattered in a cone whose axis is approximately perpendicular tothe incident laser beam) or fluorescence (light emitted from the cellsat a different wavelength from that of the incident laser beam).

Commercial hematology analyzers are intended for use on all whole bloodspecimens, including abnormal samples from patients. However, inreality, these analyzers may fail to provide accurate analyses for asmall fraction of patient samples, especially for the samples havinglyse-resistant red blood cells or fragile (lyse-sensitive) white bloodcells. For samples having lyse-resistant red blood cells, unlysed redblood cells remain following the lysis procedure and thus result in afalsely high white blood cell count, and sometimes, a falsely highhemoglobin determination. For a sample having fragile white blood cells,the white blood cell differential may not be resolved properly, and somewhite blood cells (mostly lymphocytes) are destroyed or greatlyadversely affected before the assay. Therefore, methods for adjustingthe efficacy of lysing red blood cells have remained a great challengein the field of hematology.

This invention provides a method for comprehensive analysis of blood,including complete blood count, white blood cell differential, countingreticulocytes, and counting nucleated red blood cells. No lysing agentis used to lyse red blood cells for white blood cell and hemoglobinmeasurements. The lysis-free approach simplifies the hematologyanalyzer. Such an approach requires the minimal number of reagents, andsubsequently, much fewer components for hardware and fluidics. A singlereagent could serve as both diluent and fluorescent dye, i.e., a diluentcontaining a pre-diluted fluorescent dye. Alternatively, two reagentscan be used, namely, a diluent and a concentrated fluorescent dye. Itshould be noted, however, that the method described herein can also beused when red blood cells undergo complete lysis or partial lysis.

The method described herein uses of one or more fluorescent dyes to bindand stain nucleic acids in certain blood cells (e.g., white blood cells,nucleated red blood cells, and reticulocytes) and to induce fluorescentemissions upon excitation of photons from a given source of light, suchas a laser, at an appropriate wavelength.

The method described herein uses at least one fluorescent trigger in thestep of collecting data to collect events that emit strong fluorescence,in order to separate whole blood cells and nucleated red blood cellsfrom red blood cells and platelets without the need for using a lysingagent.

The method described herein uses a plurality of optical channels and atleast one fluorescent channel for collecting data and analyzing data inorder to identify each cell population and reveal additionalinformation, such as, for example, each population of cells, includingreticulocytes, nucleated red blood cells, and major components of awhite blood cell differential analysis.

All white blood cells and nucleated red blood cells have nuclei, whichcontain a relatively high density of DNA. Immature red blood cells, orreticulocytes, contain a network of ribosomal RNA. In contrast, maturered blood cells and platelets do not contain any nucleic acids.Therefore, a fluorescent dye can be selected to differentiate twoclasses of cells, those blood cells having nucleic acids and those bloodcells not having nucleic acids. The fluorescent dye is intended topenetrate into live cells easily, bind DNA or RNA or both DNA and RNAwith high affinity, and emit strong fluorescence with adequate Stokesshift if the dye is excited by an appropriate source of light. It ispreferred that the fluorescent dye retain a significant percentage(preferably at least 10%, more preferably at least 50%) of the peakabsorption of the fluorescent dye at the wavelength of the source oflight in the visible band of the electromagnetic spectrum (approximately400 nm to 700 nm) in order to be excited properly. A plurality offluorescent dyes can also be used in the environment described herein.For example, the combination of a fluorescent dye capable ofspecifically binding to DNA and a fluorescent dye capable ofspecifically binding to RNA can be used to differentiate blood cells.

A fluorescent trigger is essential for collecting and analyzing whiteblood cells and nucleated red blood cells, both of which types of cellsare present at relatively low concentrations, as compared with red bloodcells and platelets. Mature red blood cells do not emit fluorescence oremit very weak autofluorescence. Reticulocytes emit strongerfluorescence than do mature red blood cells following RNA-dyeinteractions. Platelets in general should emit weak fluorescence,because most platelets do not contain nucleic acids. However,reticulated platelets (which contain RNA), and the possible attractionof dye molecules to surfaces of sticky platelets, may result in higherfluorescence emission on some platelets. Platelets may provide a broadrange of fluorescent signals, ranging from as low as those of mature redblood cells to as high as those of lymphocytes. A small fraction ofplatelets can show strong fluorescent signals, with the result thatthese platelets are collected in the analysis of a white blood cell datablock, because the values of the fluorescent signals from the plateletsexceed the trigger value. See, for example, FIG. 2. The cells that showmuch stronger fluorescence emission are the cells having nuclei, i.e.,all white blood cells and nucleated red blood cells. Accordingly, whiteblood cells and nucleated red blood cells can be separated from redblood cells and reticulocytes. A fluorescent trigger, usually setbetween reticulocytes and nucleated red blood cells or white bloodcells, can be used to collect events involving white blood cells andnucleated red blood cells separately for further analysis. For example,reticulocytes have maximum fluorescent signal values of 50 in thedetection system described herein. White blood cells have minimumfluorescent signal values of 150 in the detection system describedherein. The trigger can be set at a fluorescent signal value between 50and 150 in the detection system described herein. The optimal setting ofthe trigger would be 100 in the detection system described herein, butit is acceptable to set the fluorescent trigger value at, for example,75 or 125 in the detection system described herein. The scheme of thefluorescent trigger is shown in FIG. 2. Two data blocks, one data blockfor all cells (predominantly red blood cells and platelets) and theother data block for white blood cells and nucleated red blood cells,can be generated with such a setting for a fluorescent trigger. When afluorescent trigger is properly set, the hematology analyzer will onlycollect events emitting strong fluorescence, i.e., those signals fromwhite blood cells and nucleated red blood cells, and a small fraction ofbright platelets. Mature red blood cells and reticulocytes do not emitfluorescent signals of sufficient strength to be collected by thehematology analyzer. Thus, the fluorescent signals emitted by mature redblood cells and reticulocytes are not included in the second data block.

In another embodiment, all of the platelets can be stained by means ofan appropriate fluorescent dye or by means of an appropriate stainingprocedure or by means of both an appropriate fluorescent dye and anappropriate staining procedure. By these means, platelets can becompletely separated from all red blood cells, including reticulocytes,and captured as an independent population by using the fluorescentsignals of the platelets, as well as by light extinction and scatteringinformation.

In order to ensure that the fluorescence-based lysis-free method forperforming hematology analysis functions properly, the fluorescent dyemust substantially meet the following requirements:

-   -   (1) The fluorescent dye must be capable of being excited at the        appropriate wavelength. It is not necessary that the peak        absorption of the dye match exactly the wavelength of the source        of light. It is preferred that the dye retain a significant        fraction (at least about 10%, preferably at least about 50%) of        its peak absorption at the appropriate wavelength. For example,        the fluorescent dye SYTO® 41 has an absorption peak at        approximately 412 nm. This dye retains strong absorption at 405        nm, i.e., A₄₀₅/A₄₁₂ is equal to approximately 90%.    -   (2) The fluorescent dye must provide strong fluorescence        emission upon specifically binding to DNA or to RNA or to both        DNA and RNA. The fluorescent dye itself, when not bound, emits        very weak fluorescence. A fluorescent dye bound to a nucleic        acid preferably emits at least 100×, more preferably emits at        least 1000×, more fluorescence upon being excited by a source of        light. A one-dye system (for staining both DNA and RNA) or a        two-dye system (one dye for staining DNA staining, the other dye        for staining RNA) can be employed.    -   (3) The fluorescent dye must provide an adequate Stokes shift.        The fluorescence peak should differ from the absorption peak by        at least 20 nm for collecting valid signals by the fluorescence        detector (e.g., a photomultiplier tube).    -   (4) The fluorescent dye must exhibit good cell permeability. The        dye molecules are required to easily penetrate cell membranes in        order for the lysis-free assay to be effective.    -   (5) The fluorescent dye must have a high affinity for nucleic        acids and must specifically bind to nucleic acids rapidly.        Reaction kinetics should support the interaction between the dye        and the nucleic acid when the dye penetrates the membrane of the        cell.    -   (6) The fluorescent dye is preferably soluble in an aqueous        solution. The dye should not precipitate upon further dilution        thereof in aqueous solutions.    -   (7) The fluorescent dye must be stable in an aqueous solution or        in an organic solution. Stability is required for manufacturing        a dye reagent having an acceptable on-shelf life. Preferably,        the dye reagent remains stable, i.e., substantially unchanged,        for at least 12 months at ambient temperature.        The dyes suitable for use in the method described herein        substantially meet the aforementioned requirements. As mentioned        previously, there is no requirement that the fluorescent dye(s)        selected for the method described herein absorb light at any        particular wavelength or range of wavelengths or emit        fluorescence at any particular wavelength or range of        wavelengths. The following table sets forth representative        wavelengths for sources of light and fluorescent dyes that can        be used at these wavelengths.

TABLE 1 Wavelength of Category of Commercially available source of lightfluorescent examples of the fluorescent (nm) dye dye 405 Bluefluorescent SYTO ® 40 nucleic acid SYTO ® 41 stain SYTO ® 42 SYTO ® 43SYTO ® 44 SYTO ® 45 Auramine O 488 Green fluorescent SYTO ® 9 nucleicacid stain SYTO ® 10 SYTO ® 13 SYTO ® 16 SYTO ® 21 SYTO ® 23 SYTO ® 24SYTO ® 26 SYTO ® 27 SYTO ® BC Acridine orange SYBR ® 11 532 Orangefluorescent SYTO ® 80 nucleic acid stain SYTO ® 81 SYTO ® 82 SYTO ® 83SYTO ® 86 Dihydroethidium Hexidium iodide 594 Red fluorescent SYTO ® 64nucleic acid stain 633 Red fluorescent SYTO ® 17 nucleic acid stainSYTO ® 59 SYTO ® 60 SYTO ® 61 SYTO ® 62 SYTO ® 63Dyes having the trademark SYTO® are commercially available fromMolecular Probes, Inc., Eugene, Oreg. Dyes having the trademark SYBR®are commercially available from Molecular Probes, Inc., Eugene, Oreg.

The following table sets forth criteria that are suitable for selectinga source of light and ranges of wavelengths for fluorescent dyes thatare suitable for use in the method described herein.

TABLE 2 Absorption maxima Fluorescent Range of (approximate) of emissionmaxima wavelengths fluorescent (approximate) of (approximate) dye basedon fluorescent dye based on for source of wavelength of wavelength oflight (nm) source of light (nm) source of light (nm) 400-455 420-460440-490 in the presence of DNA 480-530 480-530 490-560 in the presenceof DNA, RNA, or DNA plus RNA 530-570 530-570 540-590 in the presence ofDNA 590-660 590-660 615-680 in the presence of DNAPrecise absorption maxima and fluorescent emission maxima can be foundin the following publications, all of which are incorporated herein byreference:

SYTO® Blue Fluorescent Nucleic Acid Stains, Molecular Probes ProductInformation, Revised: 15 Jan. 2001 SYTO® Green-Fluorescent Nucleic AcidStains, Invitrogen™, Molecular Probes®, Revised: 28 Apr. 2008 SYTO®Orange Fluorescent Nucleic Acid Stains, Molecular Probes ProductInformation, Revised: 13 Jan. 2001 SYTO® Red Fluorescent Nucleic AcidStains, Molecular Probes Product Information, Revised: 15 Jan. 2001

Several photodiodes or photomultiplier tubes or both photodiodes andphotomultiplier tubes can be used to detect light scattering of eachcell passing through the flow cell. Two or more photodiodes orphotomultiplier tubes can be installed for measuring axial light loss(ALL), which measures the total light lost from the laser beam at fromabout 0° to about 1° when a particle passes through the beam, andintermediate angle scatter (IAS), which measures low angle light scatter(3°-15°). Two or more photomultiplier tubes can be installed fordetecting polarized light scatter (PSS) and depolarized light scatter(DSS).

Additional photomultiplier tubes can be used to measure fluorescence atappropriate wavelengths or ranges of wavelengths, depending on thechoice of fluorescent dye(s) and the choice of wavelength of the sourceof light. Each event captured by the system described herein would thuspossess a plurality of dimensions of information, such as ALL, one ormore IAS signals, PSS, DSS, and one or more fluorescent signals. Forexample, one IAS detection channel can be used for smaller angles (i.e.,3° to 6°) and another IAS detection channel can be used for largerangles (i.e., 6° to 10°). For fluorescence detection, one detectionchannel can be used for complexes of a fluorescent dye and DNA and onedetection channel can be used for complexes of a fluorescent dye andRNA, because complexes of fluorescent dye and DNA emit a fluorescentsignal at a wavelength that is different from the wavelength of thefluorescent signal emitted by complexes of fluorescent dye and RNA. Theinformation from these detection channels can be used for furtheranalysis of the cells in a sample of blood.

The following non-limiting examples further illustrate the methoddescribed herein.

Example 1

This example illustrates the capture of white blood cells and brightplatelet events by means of a fluorescent trigger, followinginteractions of the sample of blood with dye molecules.

The following procedure, including apparatus, parameters, reagents,including diluents, was used for EXAMPLES 1-5, inclusive.

A fluorescent dye was used to stain and differentiate white blood cells(by means of DNA staining), nucleated red blood cells (by means of DNAstaining), and reticulocytes (by means of RNA staining). A fluorescenttrigger was used to generate two data blocks, thereby separating thehighly fluorescent cells (white blood cells and nucleated red bloodcells) from low fluorescent events (red blood cells and platelets). Thefirst data block was used to analyze red blood cells, platelets, andreticulocytes. The second data block was used to identify white bloodcells and nucleated red blood cells.

Each cell population was identified by means of a 407 nm flow cytometryoptical bench and four channels of scattered light: 0° (ALL), 7° (IAS),90° (PSS), and 90° depolarized (DSS). The fluorescence detector was aphotomultiplier tube capable of detecting 450 nm to 580 nm. The sourceof light is a 407 nm solid-state laser (50 mW maximum, 40 mW actuallyused). The size of the flow cell was 290 μm (length)×210 μm (width). Thelaser scanned the length (290 μm) of the flow cell. A laser rasteringsystem was used to enable high throughput (greater than 300,000 cellsper second) under lysis-free conditions. See U.S. patent applicationSer. No. 12/767,611, filed Apr. 26, 2010, and entitled METHOD FORDISCRIMINATING RED BLOOD CELLS FROM WHITE BLOOD CELLS BY USING FORWARDSCATTERING FROM A LASER IN AN AUTOMATED HEMATOLOGY ANALYZER, previouslyincorporated herein by reference, for details of the laser rasteringsystem. However, a laser rastering system is not required to carry outthe method described herein. Similarly, a 407 nm flow cytometry opticalbench is not required to carry out the method described herein. Otherwavelengths, other lasers, and other sources of light can be used tocarry out the method described herein.

A fresh sample of blood was diluted to a concentration of 1 part bloodper 50 parts diluent to serve as the reference and calibration standard.The diluted sample of blood, at a red blood cell concentration ofapproximately 0.1 million red blood cells/μL, should exhibit little orno coincidences on the flow cytometer of the hematology analyzer. Threeto six runs were conducted with the CELL-DYN® Sapphire™ hematologyanalyzer and three to six runs were conducted under the lysis-freeconditions described herein. For EXAMPLES 1-5, inclusive:

-   -   (1) Flow rate (red blood cells)=Flow rate (total blood cells)        times percentage of red blood cells, where percentage of red        blood cells equals the fraction of events attributed to red        blood cells.    -   (2) Calibration factor=Flow rate (red blood cells) divided by        concentration of red blood cells    -   (3) Cell count=Flow rate (sample) divided by the calibration        factor    -   (4) The diluent was CELL-DYN® Sapphire™ diluent/sheath    -   (5) The fluorescent dye was SYTO® 41, 17 micromolar,        commercially available from Molecular probes, Inc., Eugene Oreg.    -   (6) The ratio of the sample of blood to the dye reagent to the        diluent/sheath was approximately 1 part by volume sample of        blood to 7.5 parts by volume dye reagent to 77.5 parts by volume        diluent/sheath.    -   (7) Duration of incubation was 30 seconds at a temperature of        about 41° C. (±1° C.) to allow sufficient staining of cells        having nucleic acid content, i.e., white blood cells, nucleated        red blood cells, and reticulocytes.    -   (8) Conditions for acquisition of data:        -   (A) Red blood cell file (0.2 second, IAS trigger=75, for            analysis of red blood cells, reticulocytes, and platelets)        -   (B) White blood cell file (20 seconds, FL trigger=75, for            analysis of white blood cells, white blood cell            differential, and nucleated red blood cells)

The measurement process was initiated immediately following incubationof the sample. The cell stream, surrounded by the sheath solution, wasintroduced to the flow cell. The flow velocity of the sheath was 6m/sec, which resulted in approximately 300,000 red blood cell eventspassing through the flow cell per second (for a blood sample having5×10⁶ red blood cells/μL). A laser rastering system, described in U.S.patent application Ser. No. 12/767,611, filed Apr. 26, 2010, andentitled METHOD FOR DISCRIMINATING RED BLOOD CELLS FROM WHITE BLOODCELLS BY USING FORWARD SCATTERING FROM A LASER IN AN AUTOMATEDHEMATOLOGY ANALYZER, previously incorporated herein by reference, wasused to scan the stream of cells. The spot size of the laser beam was 20μm×10 μm. The size of the sample core stream is typically 65 μm(width)×19 μm (depth).

The data, including the light scattering signals (ALL, IAS, PSS and DSS)and fluorescence signals (FL), were collected for each captured event. Adata block, triggered by IAS, was collected in 0.2 second for allcellular particles. Electronic noises were minimized by the IAS trigger.57,649 red blood cell events, equivalent to a red blood cell count of4.4×10⁶ red blood cells/μL without coincidence correction, and 2,082 PLTevents, equivalent to a platelet count of 157×10³ platelets/μL withoutcoincidence correction, were captured for a whole blood specimen having4.8×10⁶ red blood cells/μL and 218×10³ platelets/μL PLT (referencevalues were measured by a CELL-DYN® Sapphire™ hematology analyzer). Thediscrepancies were a result of the approximately 10% coincidencesinvolving red blood cells only and the approximately 20-30% coincidencesinvolving red blood cells and platelets observed for all of the samplesof blood. Reticulocytes (2.5%) were also quantified by usingfluorescence in the same runs (% Reticulocytes=3.1% as referenced by theCELL-DYN® Sapphire™ hematology analyzer).

The cytogram of FIG. 3 illustrates the separation of populations ofmajor cells of a sample of whole blood, i.e., red blood cells,platelets, and white blood cells, by means of axial light loss (ALL) andintermediate angle scatter (IAS) of light.

The cytogram of FIG. 4 illustrates the further separation ofreticulocytes from mature red blood cells by means of fluorescence,following interaction of a fluorescent dye and RNA.

Example 2

This example illustrates that the method described herein separates redblood cells and platelets from other components of a sample of blood.The sample set included 103 specimens of whole blood, namely, 35 normalsamples, 68 abnormal samples, with 12 samples containing or suspected ofcontaining nucleated red blood cells. Two runs were carried out for eachsample of blood; CELL-DYN® Sapphire™ hematology analyzer was used as theprimary reference, i.e., to determine to what degree the methoddescribed herein compares with a hematology analyzer that requires alysing agent.

The apparatus, diluent, fluorescent dye, preparation of the sample, andconditions for measurement were the same as that described in EXAMPLE 1.

The plot in FIG. 5 illustrates the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for counting red blood cells. The slope of the bestlinear fit was 0.899 (R²=0.933). The approximately 10% bias was due tocoincidences between red blood cells in the runs.

The plot in FIG. 6 illustrates the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for counting platelets. The slope of the best linearfit was 0.783 (R²=0.971). The approximately 20% to 30% bias was due tocoincidences between red blood cells and platelets in the runs.

Example 3

This example illustrates the analysis of a white blood celldifferential. The apparatus, diluent, fluorescent dye, preparation ofthe sample, and conditions for measurement were the same as thatdescribed in EXAMPLE 1.

The data, triggered by fluorescent signals (FL), were collected in 20seconds for all events having a fluorescent signal (FL) higher than 75,including all white blood cells, nucleated red blood cells, if therewere any, and a small fraction of bright platelets. 8586 white bloodcell events, equivalent to a white blood cell count of 6.48×10³ whiteblood cells/μL, were captured for a specimen of whole blood having awhite blood cell count of 6.29×10³ white blood cells/μL (based on thereference value from a CELL-DYN® Sapphire™ hematology analyzer). Thesubpopulations of white blood cells, including neutrophils, lymphocytes,monocytes, and eosinophils, were analyzed by means of the signalsobtained from a plurality of optical channels.

The cytogram of FIG. 7 illustrates separation of white blood cells fromfluorescent (bright) platelets of a sample of whole blood by means ofaxial light loss (ALL) and intermediate angle scatter (IAS) of light.

The cytogram of FIG. 8 illustrates the three major subpopulations ofwhite blood cells, namely, neutrophils (NE), lymphocytes (LY), andmonocytes (MO) by means of polarized side scatter (PSS) and axial lightloss (ALL) of light.

The cytogram of FIG. 9 illustrates the three major subpopulations ofwhite blood cells, namely, neutrophils (NE), lymphocytes (LY), andmonocytes (MO) by means of polarized side scatter (PSS) and axial lightloss (ALL) of light.

The cytogram of FIG. 10 illustrates the separation of eosinophils (EO)from the remaining white blood cells by means of depolarized sidescatter (DSS) and polarized side scatter (PSS) of light.

The values of white blood cell differential are listed in the followingtable.

TABLE 3 Amount of component as Amount of component as determined bydetermined by method of White blood cell CELL-DYN ® Sapphire ™ Example 3subpopulation* hematology analyzer (%) (lysis-free assay) (%)Neutrophils (NE) 61 59 Lymphocytes (LY) 21 24 Monocytes (MO) 15 13Eosinophils (EO)  3  4 *Basophils were not reported on account oflimitations of the optical channels.

Example 4

This example illustrates how well the method described herein correlateswith a CELL-DYN® Sapphire™ hematology analyzer for the analysis of awhite blood cell differential. The apparatus, diluent, fluorescent dye,preparation of the sample, and conditions for measurement were the sameas that described in EXAMPLE 1.

The data, triggered by fluorescent signals (FL), were collected in 20seconds for all events having a fluorescent signal (FL) higher than 75,including all white blood cells, nucleated red blood cells, if therewere any, and a small fraction of bright platelets. 8586 white bloodcell events, equivalent to a white blood cell count of 6.48×10³ whiteblood cells/4, were captured for a specimen of whole blood having awhite blood cell count of 6.29×10³ white blood cells/4 (based on thereference value from a CELL-DYN® Sapphire™ hematology analyzer). Thesubpopulations of white blood cells, including neutrophils, lymphocytes,monocytes, and eosinophils, were analyzed by means of the signalsobtained from a plurality of optical channels.

The plot in FIG. 11 illustrates the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for white blood cells. The slope of the best linear fitwas 1.019 (R²=0.988).

The plot in FIG. 12 illustrates the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for neutrophils. The slope of the best linear fit was1.024 (R²=0.979).

The plot in FIG. 13 illustrates the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for lymphocytes. The slope of the best linear fit was0.967 (R²=0.983).

The plot in FIG. 14 illustrates the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for monocytes. The slope of the best linear fit was0.959 (R²=0.890).

The plot in FIG. 15 illustrates the correlation between the CELL-DYN®Sapphire™ hematology analyzer and a hematology analyzer using the methoddescribed herein for eosinophils. The slope of the best linear fit was0.994 (R²=0.982).

Example 5

This example illustrates the counting of nucleated red blood cells bythe method described herein. The apparatus, diluent, fluorescent dye,preparation of the sample, and conditions for measurement were the sameas that described in EXAMPLE 1.

The data, triggered by fluorescence signals (FL), were collected in 20seconds for all events having a fluorescent signal (FL) higher than 75,including all white blood cells, nucleated red blood cells, if therewere any, and a small fraction of bright platelets. 8586 white bloodcell events, equivalent to a white blood cell count of 6.48×10³ whiteblood cells/μL, were captured for a specimen of whole blood having awhite blood cell count of 6.29×10³ white blood cells/μL (based on thereference value from a CELL-DYN® Sapphire™ hematology analyzer). Thesubpopulations of white blood cells, including neutrophils, lymphocytes,monocytes, and eosinophils, were analyzed by means of the signalsobtained from a plurality of optical channels.

A sample of whole blood having a confirmed content of nucleated redblood cells (nucleated red blood cells/white blood cells=3.99% asmeasured by a CELL-DYN® Sapphire™ hematology apparatus) was analyzed todetermine the concentration of nucleated red blood cells according tothe method described herein.

The cytogram in FIG. 16 illustrates nucleated red blood cell events as aseparate population from the white blood cells. The cytogram is a plotof polarized side scatter (PSS) against axial light loss (ALL) of light.The ratio of nucleated red blood cells to white blood cells wascalculated to be 4.5%.

Example 6

This example illustrates the use of a source of light different fromthat used in EXAMPLES 1-5, inclusive, and a fluorescent dye differentfrom that used in EXAMPLES 1-5, inclusive, for an analysis of a whiteblood cell differential. The following procedure, including apparatus,parameters, reagents, including diluents, was used for Example 6.

This example illustrates capturing of white blood cells and brightplatelet events by means of a fluorescent trigger, followinginteractions of the sample with dye molecules.

A fluorescent dye, SYBR® 11, commercially available from Molecularprobes, Inc., Eugene Oreg., was used to stain and differentiate whiteblood cells (DNA), nucleated red blood cells (DNA), and reticulocytes(RNA). The data block thus collected was used to identify white bloodcells and nucleated red blood cells.

Each cell population was identified by means of a 488 nm flow cytometryoptical bench and four channels of scattered light: 0° (ALL), 7° (IAS),and 90° (PSS). The 488 nm source of light was a solid-state diode laser(20 mW). The size of the flow cell was 400 μm (length)×160 μm (width).The laser scanned the length (400 μm) of the flow cell. The detectiondevices included a plurality of optical channels, including ALL, IAS,and PSS, and a photomultiplier tube detector for fluorescence (FL1, 515nm to 545 nm).

Preparation of the sample involved a single dilution of the sample ofblood with the fluorescent dye and the diluent/sheath. The dilutionratio was approximately 1 part by volume sample of blood to 3.2 parts byvolume fluorescent dye reagent to 31 parts by volume diluent/sheath. Thediluted sample of blood was incubated at a temperature of 40° C. (±1°C.) for 32 seconds to allow sufficient staining of cells having acontent of nucleic acids, i.e., white blood cells, nucleic red bloodcells, and reticulocytes.

The measurement process was initiated immediately following incubationof the sample. The cell stream, surrounded by the sheath solution, wasintroduced to the flow cell. The flow velocity of the sheath flow was 6m/s, which resulted in approximately 200 white blood cell events passingthrough the flow cell per second (for a sample of blood having 5×10³white blood cells/μL). The spot size of the laser beam was 65 μm×17 μm.The size of the sample core stream is typically 70 μm (width)×5 μm(depth). The total measurement time was approximately nine (9) seconds.

The data, including the light scattering signals (ALL, IAS, and PSS) andfluorescence signals (FL1), were collected for each event triggered byfluorescence. 5760 white blood cell events were captured for a wholeblood specimen having 16×10³ white blood cells. The subpopulations ofwhite blood cells, including neutrophils, lymphocytes, and monocytes,were differentiated by means of cytograms. The cytogram of FIG. 17illustrates white blood cell differentiation of a sample of whole bloodby means of the method described herein.

The values of white blood cell differential are listed in the followingtable.

TABLE 4 Amount of component as determined by Amount of component asWhite blood cell CELL-DYN ® Sapphire ™ determined by method ofsubpopulation* hematology analyzer (%) Example 6 (%) Neutrophils 86 88Lymphocytes  4  3 Monocytes  9  9 *Eosinophils and basophils were notreported on account of limitations of the optical channels.

Various modifications and alterations of this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention, and it should be understood that thisinvention is not to be unduly limited to the illustrative embodimentsset forth herein.

1-24. (canceled)
 25. An automated hematology analyzer for counting cellsin a sample of whole blood, said analyzer comprising: an excitationsource positioned to excite particles within the blood sample; and aplurality of detectors including an axial light loss detector positionedto measure axial light loss from the excited blood sample, anintermediate angle scatter detector positioned to measure intermediateangle scatter from the excited blood sample, a side scatter detectorpositioned to measure 90° side scatter from the excited blood sample,and a fluorescence detector positioned to measure fluorescence emittedfrom the excited blood sample, wherein the automated hematology analyzeris configured to: dilute a sample of whole blood with a diluent, whereinthe sample comprises a plurality of white blood cells, nucleated redblood cells, red blood cells, platelets, and reticulocytes; contact thesample with at least one fluorescent dye that specifically binds to andstains one or more nucleic acids in the white blood cells, the nucleatedred blood cells, and/or the reticulocytes in the sample; generate aplurality of events, wherein each event comprises a plurality of lightscattering signals and a fluorescence emission signal generated by acell in the sample; separate the events into two data blocks using afluorescent trigger, wherein the first data block includes events havinga fluorescence emission signal that is below the fluorescent trigger,and the second data block includes events having a fluorescence emissionsignal that is above the fluorescent trigger; analyze the events in thefirst data block to count the number of red blood cells, platelets, andreticulocytes in the sample; and analyze the events in the second datablock to count the number of white blood cells and nucleated red bloodcells in the sample.
 26. The automated hematology analyzer of claim 25,wherein the at least one fluorescent dye specifically binds to andstains a nucleic acid in the reticulocytes.
 27. The automated hematologyanalyzer of claim 25, wherein the source of light is a laser.
 28. Theautomated hematology analyzer of claim 25, wherein the source of lighthas a wavelength of from 350 nm to 700 nm.
 29. The automated hematologyanalyzer of claim 28, wherein the source of light has a wavelength offrom 400 nm to 455 nm.
 30. The automated hematology analyzer of claim28, wherein the source of light has a wavelength of from 480 nm to 530nm.
 31. The automated hematology analyzer of claim 28, wherein thesource of light has a wavelength of from 530 nm to 570 nm.
 32. Theautomated hematology analyzer of claim 28, wherein the source of lighthas a wavelength of from 590 nm to 660 nm.
 33. The automated hematologyanalyzer of claim 25, wherein the at least one fluorescent dye has anabsorption maximum of from 420 nm to 460 nm.
 34. The automatedhematology analyzer of claim 25, wherein the at least one fluorescentdye has an absorption maximum of from 480 nm to 530 nm.
 35. Theautomated hematology analyzer of claim 25, wherein the at least onefluorescent dye has an absorption maximum of from 530 nm to
 570. 36. Theautomated hematology analyzer of claim 25, wherein the at least onefluorescent dye has an absorption maximum of from 590 to 660 nm.
 37. Theautomated hematology analyzer of claim 25, wherein the at least onefluorescent dye has an emission maximum of from 440 nm to 490 nm in thepresence of DNA.
 38. The automated hematology analyzer of claim 25,wherein the at least one fluorescent dye has an emission maximum of from490 nm to 560 nm in the presence of DNA or RNA or both DNA and RNA. 39.The automated hematology analyzer of claim 25, wherein the at least onefluorescent dye has an emission maximum of from 540 nm to 590 nm in thepresence of DNA.
 40. The automated hematology analyzer of claim 25,wherein the at least one fluorescent dye has an emission maximum of from615 nm to 680 nm in the presence of DNA.
 41. The automated hematologyanalyzer of claim 25, wherein the hematology analyzer is configured toanalyze the events in the second data block to count the number of cellsin each of a plurality of white blood cell subpopulations.
 42. Theautomated hematology analyzer of claim 25, wherein the analyzer employsno more than two reagents, said reagents being a diluent and afluorescent dye.
 43. The automated hematology analyzer of claim 34,wherein contacting the sample with at least one fluorescent dyecomprises diluting the sample of whole blood in a diluent in which thefluorescent dye is pre-diluted.
 44. The automated hematology analyzer ofclaim 34, wherein contacting the sample with at least one fluorescentdye occurs after diluting the sample of whole blood with the diluent.45. The automated hematology analyzer of claim 25, wherein thehematology analyzer is configured to contact the sample with afluorescent dye and/or stain that fluorescently labels platelets in thesample, and analyze events from the second data block to count thenumber of platelets in the sample.